The activated newborn neurons participate in enriched environment induced improvement of locomotor function in APP/PS1 mice

2.1 Animals, EE, and EdU administration

APP/PS1 mice (Thy1–APPKM670/671Nl, Thy1–PS1 L166P) were kindly provided by Mathias Jucker (Tubingen University). The generation and characterization of APP/PS1 mice has been described previously (Pentkowski et al., 2018). At the age of three weeks, 16 male wild-type mice on a 129/Sv background and 18 male APP/PS1 mice were intraperitoneally injected with 100 mg/kg of EdU once a day for 7 days (ThermoFisher Scientific) and were then randomly assigned to either SE or EE housing conditions for 1 month. Standard laboratory cages (33 cm × 18 cm × 14 cm) were used for SE, whereas larger cages (55 cm × 34 cm × 20 cm) with various toys were used for the EE condition (Hu et al., 2010). In the EE cages, the animals also had access to a variety of different objects, such as running wheels, colored tunnels, and visually stimulating toys, which were changed weekly. All animals were tested on the elevated plus maze (EPM) and a fear conditioning task after 1 month of experience with SE or EE.

All mice were housed in conditions of constant temperature (20–22°C) and a 12-hr light–dark cycle with access to food and water ad libitum. All animal experiments were performed under an animal study protocol approved by the ethics committee of Shanghai Jiao Tong University School of Medicine.

2.2 EPM

All tests were conducted according to a previous study (Zhu et al., 2016). After EE or SE exposure, the mice were habituated to handling and were transported from the colony room to the behavioral room for 3 days before behavioral tests were performed. The mice were given 1 hr to habituate after transport to the behavioral room before any tests were conducted. All apparatuses and testing chambers were cleaned with 75% ethyl alcohol wipes between animals. The EPM apparatus was made of dark gray plastic and consisted of two open arms (30 × 7 × 0.25 cm) opposing two enclosed arms (30 × 7 × 15 cm) elevated 60 cm from the floor. The animals were placed in the central area of the apparatus with their head facing an enclosed arm (test duration: 5 min). The test was performed in a sound-attenuated and temperature-controlled (22 ± 1°C) room illuminated by one 40-W fluorescent bulb placed 3 m above the apparatus. Digitized video recordings (30 frames per s) obtained by EthoVision software (Noldus Information Technology, Leesburg, VA) were used for behavioral analysis. The time spent in the open arms and the frequency of entry into the open arms were used as innate behavioral indexes.

2.3 Contextual fear conditioning

The contextual fear conditioning test was performed using a UGO Basile Fear Conditioning System (Model 46850). During the training session, each mouse was placed in a shock chamber for 3 min. Black-and-white striped wallpaper applied to the sides of the experimental cage, tactile sensations on the feet of the animals, and the odor of alcohol formed the contextual information for context A of fear conditioning training. During the training session, the mice were first acclimated to the experimental cage for 3 min and then exposed to by a paired voice (4 kHz, 76 db, duration of 30 s) and plantar electric shock (0.85 mA, 2 s). After the plantar electric shock, the mice stayed in the experimental cage for an additional 30 s. At the end of the experiment, the mice were returned to the breeding cage. After each mouse experiment, the test chamber was wiped with 75% ethanol. Twenty-four hours later, the animals were subjected to fear conditioning test A (fear A). First, each mouse was placed in the context A environment, and no sound stimulation was given. The recording was performed for 3 min. After each mouse experiment, the test chamber was wiped with 75% ethanol. Three hours later, the experimental mice were then subjected to fear conditioning test B (fear B). The sides of the chamber were covered with light gray wallpaper, the bottom was covered with a smooth gray plastic plate, and the experimental cage was wiped with 4% acetic acid solution instead of 75% ethanol (context B). During the experiment, the animals first adapted to the new environment for 3 min, and then received a plantar electric shock once. Recordings continued for 2 min after the plantar electric shock. After each mouse was tested, the chamber was wiped with 4% acetic acid. The freezing times in the fear A and fear B tests were recorded as the cognitive index.

2.4 Immunofluorescence

For immunofluorescence, coronal brain slices were sectioned at a thickness of 30 μm, washed with phosphate buffered saline (PBS), and then incubated for blocking with permeabilization buffer (0.3% Triton-100 in PBS) containing 10% donkey serum for 45–60 min. The sections were incubated in primary antibodies against NeuN (Millipore) and c-Fos (Cell Signaling Technology) overnight at 4°C. The sections were rinsed three times (10 min each) in PBS, permeabilized with 0.1% Tween-20 in PBS, and then incubated at 4°C overnight with Alexa Fluor secondary antibodies specific for the primary antibody host. Following incubation with secondary antibodies, the sections were rinsed three times (10 min each) in PBS, mounted on gelatin-coated glass slides, and coverslipped using mounting medium. For EdU staining, we used Click-iT^® Plus EdU Imaging Kits (Life Technologies). The images were obtained using an Olympus microscope. The analysis of immuno-positive neurons in the hippocampal area was quantified with ImageJ software. Five brain sections were collected for quantifying the positive cells in each mouse.

2.5 Statistical analysis

The statistical analysis was performed using SPSS 16.0 software. Two-way ANOVA, one-way ANOVA, and Student's t test were performed for the data analysis. p < 0.05 was considered statistically significant.

3.1 EE improved the behavioral performance of APP/PS1 mice

After 1 month of SE exposure, the mice were tested in the EPM. The results showed that there were no differences between the APP/PS1 mice and WT mice in the frequency of entry into or the time spent in the open arms (time: WT + SE, 39.17 ± 9.00 s; APP/PS1 + SE, 44.82 ± 8.73 s; p = 0.718; frequency: WT + SE, 15.63 ± 1.97; APP/PS1 + SE, 15.67 ± 2.27; p = 0.994; Figure 1a). After 1 month of exposure to EE, the time spent in and the frequency of entry into the open arms were significantly increased in the APP/PS1 mice (time: APP/PS1 + SE, 44.82 ± 8.73 s; APP/PS1 + EE, 81.96 ± 15.02 s; p = 0.046; frequency: APP/PS1 + SE, 15.67 ± 2.27; APP/PS1 + EE, 38.44 ± 4.74; p = 0.004; Figure 1a), suggesting improved locomotor accompanied by less anxiety in the EE-exposed APP/PS1 mice. However, the time spent in and the frequency of entry into the open arms did not significantly change between the SE- and EE-exposed WT mice (time: WT + SE, 39.17 ± 9.00 s; WT + EE, 49.65 ± 8.73 s; p = 0.516; frequency: WT + SE, 15.63 ± 1.97; WT + EE, 22.38 ± 4.53; p = 0.212; Figure 1a).

The mice then performed a fear conditioning task. The mice were placed into the chamber, and the freezing times were measured in both the fear A and fear B stages. The results showed that there were no significant differences in the cognitive index between these groups (time: WT + SE, 46.58 ± 2.78 s; WT + EE, 48.23 ± 1.50 s; APP/PS1 + SE, 46.33 ± 2.71 s; APP/PS1 + EE, 49.46 ± 3.27 s; p = 0.821; frequency: WT + SE, 121.49 ± 9.52; WT + EE, 119.58 ± 9.65; APP/PS1 + SE, 118.77 ± 11.73; APP/PS1 + EE, 124.39 ± 10.40; p = 0.981; Figure 1b).

3.2 EE increased the number of c-Fos-positive neurons in the DG area but not in CA1 and CA3

The behavioral trials were then followed by the measurement of c-Fos expression, which indicates the extent of neuronal activation, in the hippocampus. Consistent with the behavior performance on the EPM, a dramatic increase in the number of c-Fos-positive cells was observed in DG area of the EE-exposed APP/PS1 mice (DG: WT + SE, 180.58 ± 12.27; WT + EE, 235.82 ± 27.56; APP/PS1 + SE, 285.77 ± 22.96; APP/PS1 + EE, 449.04 ± 105.03; Figure 2). However, in the brain regions of CA1 and CA3, an increase in c-Fos-positive neurons was observed in the APP/PS1 mice compared with the WT mice, and the number of c-Fos-positive neurons was not affected by EE exposure in either the WT or APP/PS1 mice (CA1: WT + SE, 289.80 ± 52.50; WT + EE, 294.16 ± 33.44; APP/PS1 + SE, 615.18 ± 68.81; APP/PS1 + EE, 607.05 ± 93.26; CA3: WT + SE, 281.98 ± 31.01; WT + EE, 295.23 ± 23.14; APP/PS1 + SE, 467.73 ± 20.55; APP/PS1 + EE, 480.92 ± 41.51; Figure 2).

3.3 EE increased hippocampal neurogenesis and activated c-Fos expression in newborn cells in APP/PS1 mice

The results showed that, compared with the WT mice exposed to SE, the number of newborn neurons decreased in the APP/PS1 mice. However, that number was significantly increased in the APP/PS1 mice under EE conditions but not in the WT mice exposed to EE (neuronal EdU: WT + SE, 117.67 ± 0.67; WT + EE, 128.00 ± 4.04; APP/PS1 + SE, 80.33 ± 3.84; APP/PS1 + EE, 109.00 ± 1.73; total Edu: WT + SE, 166.67 ± 5.24; WT + EE, 179.67 ± 2.91; APP/PS1 + SE, 139.00 ± 2.08; APP/PS1 + EE, 160.33 ± 6.06; the ratio of neuronal EdU to total Edu: WT + SE, 0.71 ± 0.03; WT + EE, 0.71 ± 0.02; APP/PS1 + SE, 0.58 ± 0.02; APP/PS1 + EE, 0.68 ± 0.04; Figure 3). The results of c-Fos, EdU, and NeuN staining indicated that in the EE-exposed APP/PS1 mice, c-Fos was expressed in newborn neurons (Figure 4), while there was no expression of c-Fos in newborn neurons in the other three groups (Figure S1).